Media Information
Back to Microbiology Laboratory Actinomycete Isolation Agar Actinomycetes are gram-positive bacteria, which show marked chemical and morphological diversity but form a distinct evolutionary line of organisms that range from coccoid and pleomorphic forms to branched filaments. Actinomycetes form an integral part of soil, water and vegetation. Actinomycete development leads to the formation of volatile metabolites. Traces of these volatile metabolites are sufficient to impart disagreeable odor to water or a muddy flavor to fish. Actinomycetes also cause disruptions in wastewater treatment by forming massive growths, which are capable of producing thick foam in the activated sludge process. Actinomyces Isolation Agar used for isolation and propagation of Actinomycetes from soil and water was formulated by Olsen. Actinomycete Isolation Agar contains sodium caseinate as nitrogen source. Asparagine in addition to being an amino acid is also a source of nitrogen. Sodium propionate is used as a substrate in anaerobic fermentation. Dipotassium phosphate provides the buffering system. The sulphates serve as source of Sulphur and metallic ions. Glycerol serves as an additional source of carbon. Inoculate the plates with 1 drop of diluted culture or specimen and spread over the surface using a sterile bent glass rod. Incubate at 35-37°C for 40-72 hours. The media can be used for long term storage after sufficient growth is obtained. Agar slants are used for maintenance of cultures over a shorter period of time. Blood Agar Brain Heart Infusion Brain heart infusion (BHI) is a growth medium for growing microorganisms. It is a nutrient-rich medium, and can therefore be used to culture a variety of fastidious organisms. In particular, it has been used to culture streptococci, pneumococci and meningococcal, which can be otherwise challenging to grow. BHI is made by combining an infusion from boiled bovine or porcine heart and brain with a variety of other nutrients. BHI broth is often used in food safety, water safety, and antibiotic sensitivity tests. The earliest version of brain heart infusion media was made in 1919 when Edward Rosenow combined dextrose broth with calf brain tissue to grow streptococci. This was modified in 1923 by Russell Haden while working on dental pathogens. Modern BHI typically uses an infusion from porcine brains and hearts rather than calf brain tissue, and uses disodium phosphate as a buffer, rather than the calcium carbonate used by Rosenow and Haden. BHI typically contains infusion of beef or pig heart as well as calf brain, a source of amino acids (often either digested gelatin or other animal tissue), salt, disodium phosphate as a buffer, and glucose as a source of sugar. Many formulations for BHI agar also exist, in which agar is added as a gelling agent for growing plates of microorganisms. Brain-Heart Infusion Broth can be supplemented with antibiotics, varying amounts of sodium chloride, yeast extract, and serum to provide a rich medium for bacteria, yeasts and pathogenic fungi.3 The addition of 0.1% agar can be used to lower oxygen tension, providing an atmosphere to support the growth of aerobic, microaerophilic, and obligate anaerobic microorganisms. Brilliant Green Bile Broth Brilliant Green Bile Broth 2% is one of the most widely used medium for the detection of coliform bacteria in water, wastewater, foods, and milk and dairy products. This medium is formulated as per APHA for the presumptive identification and confirmation of coliform bacteria. This medium is also recommended by the ISO Committee for enumeration of coliforms by most probable number technique. Peptone serves as a source of essential nutrients. Lactose is the fermentable carbohydrate. Bile inhibits gram-positive bacteria whereas the gram-negative bacteria are inhibited by brilliant green. Production of gas from lactose fermentation is detected by incorporating inverted Durham's tube, which indicates the positive evidence of fecal coliform since non fecal coliforms growing in this medium do not produce gas. Further gas production in EC broth (M127) at 45°C used as a confirmation of fecal coliform. Gram-positive spore formers may produce gas if the bile or brilliant green inhibition is weakened by reaction with food material. During examination of water samples, growth from presumptive positive tubes showing gas in Lactose Broth (M026) or Lauryl Tryptose Broth (M080) is inoculated in Brilliant Green Bile Broth 2% (M121). Gas formation within 48 ± 2 hours confirms the presumptive test). Brilliant Green Bile Broth 2% is used for the detection of coliform bacteria in water, food, and dairy products in a laboratory setting. Brilliant Green Bile Broth 2% is not intended for use in the diagnosis of disease or other conditions in humans. The coliform group of bacteria includes aerobic and facultative anaerobic, Gram-negative, non-spore-forming bacilli that ferment lactose and form acid and gas at 35°C within 48 hours. Members of the Enterobacteriacae comprise the majority of this group, but organisms such as Aeromonas spp. may also be included. Procedures to detect and confirm coliforms are used in testing water, foods, dairy products and other materials. Brilliant Green Bile Broth 2% is used to confirm a positive presumptive test result. The formulation for Brilliant Green Bile Broth was developed by the Association of Official Analytical Communities (AOAC) and the American Public Health Association (APHA). The basal medium is composed of peptone, which contains 2% bile, and brilliant green dye. Bile is inhibitory to gram-positive microorganisms, while brilliant green dye inhibits selected gram-negative bacilli. Lactose-fermenting organisms resistant to these inhibitors are detected by the production of gas. Gas production is noted by the appearance of bubbles in the Durham tube. Lactose Broth Examination of water, foods, ingredients and raw materials, for the presence of marker groups such as coliforms is one of the most common tests in a microbiology laboratory, partly because of the relative ease and speed with which these tests can be accomplished. Where it is claimed that drinking water has been processed for safety, the finding of such organism demonstrates a failure of the process. It is a valuable bacterial indicator for determining the extent of fecal contamination of recreational surface waters or drinking water. Lactose Broth is recommended by APHA in the performance and confirmation of the presumptive test for coliform bacteria in water, food and milk. This medium was initially listed as an alternative to Lauryl Sulfate Broth in the presumptive Standard Total Coliform Multiple-Tube (MPN) Test for water analysis. Although it is not the original formulation, Lactose Broth provides excellent results in Eijkman Assays of gas production at 45°C, which is a characteristic of Escherichia coli. While preparing this medium it is important to avoid overheating and to distribute it into tubes before sterilization. Peptic digest of animal tissue and beef extract in the medium supply essential nutrients to the organisms. Lactose is a fermentable carbohydrate for the coliforms. Tubes of Lactose Broth are inoculated with dilutions of water or milk, etc. under test, and incubated at 35°C and examined for gas formation after 24 and 48 hours. Members of the coliform group are defined as aerobic and facultative anaerobic gram-negative and non-sporing bacilli, which ferment lactose with gas formation within 48 hours at 35°C. In testing dairy products, Lactose Broth is used only in the completed test (3). Large water samples may require double strength Lactose Broth to minimize the final volume LB Broth Lysogeny broth (LB), a nutritionally rich medium, is primarily used for the growth of bacteria. The initialization is also commonly, albeit incorrectly, taken to mean Luria broth, Lennox broth, or Luria-Bertani medium. According to its creator Giuseppe Bertani, the abbreviation LB was actually intended to stand for lysogeny broth.1 The formula of the LB medium was published in 1951 in the first paper of Bertani on lysogeny. In this article he described the modified single-burst experiment and the isolation of the phages P1, P2, and P3. He had developed the LB medium to optimize Shigella growth and plaque formation. LB media formulations have been an industry standard for the cultivation of Escherichia coli as far back as the 1950s. These media have been widely used in molecular microbiology applications for the preparation of plasmid DNA and recombinant proteins. It continues to be one of the most common media used for maintaining and cultivating laboratory recombinant strains of Escherichia coli.8 For physiological studies however, the use of LB medium is to be discouraged: Hiroshi Nikaido describes the limitations of peptides and amino acids as nutrients (with major switches in early log phase) and limited availability of Mg and Ca in this important commentary, available online. There are several common formulations of LB. Although they are different, they generally share a somewhat similar composition of ingredients used to promote growth, including the following: * Peptides and casein peptones * Vitamins (including B vitamins) * Trace elements (e.g. nitrogen, sulfur, magnesium) * Minerals Sodium ions for transport and osmotic balance are provided by sodium chloride. Tryptone is used to provide essential amino acids such as peptides and peptones to the growing bacteria, while the yeast extract is used to provide a plethora of organic compounds helpful for bacterial growth. These compounds include vitamins and certain trace elements. MacConkey Agar Selective: It contains bile salts (to inhibit most Gram-positive bacteria), crystal violet dye (which also inhibits certain Gram-positive bacteria) Differential: neutral red dye (which turns pink if the microbes are fermenting lactose). MacConkey agar is an indicator, a selective and differential culture medium for bacteria designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacilli and differentiate them based on lactose fermentation. The crystal violet and bile salts inhibit the growth of gram-positive organisms which allows for the selection and isolation of gram-negative bacteria. Enteric bacteria that have the ability to ferment lactose can be detected using the carbohydrate lactose, and the pH indicator neutral red. Composition: * Peptone – 17 g * Proteose peptone – 3 g * Lactose – 10 g * Bile salts – 1.5 g * Sodium chloride – 5 g * Neutral red – 0.03 g * Crystal violet – 0.001 g * Agar – 13.5 g * Water – add to make 1 litre; adjust pH to 7.1 +/− 0.2 Mannitol Salt Agar A. B. C. D. A. Gram + Staphylococcus: fermenting mannitol: medium turns yellow (e.g. S. aureus) B.Gram + Staphylococcus: not fermenting mannitol, medium does not change color (e.g. S. epidermidis) C.Gram + Streptococcus: inhibited growth D. Gram -: inhibited growth Selective: 7.5% NaCl for selectivity of halotolerant species of Staphylococcus Differential: Phenol Red pH indicator: Yellow below pH=6.8, Red @ pH= 7.2-8.4, Pink @ pH=8.4 or above | Mannitol fermentation vs. non-fermenting. Mannitol salt agar or MSA is a commonly used selective and differential growth medium in microbiology. It encourages the growth of a group of certain bacteria while inhibiting the growth of others. This medium is important in medical laboratories as one method of distinguishing pathogenic microbes in a short period of time. It contains a high concentration (about 7.5%-10%) of salt (NaCl), making it selective for Gram-positive bacteria (Staphylococcus and Micrococcaceae) since this level of salt is inhibitory to most other bacteria It is also a differential medium for mannitol-fermenting staphylococci, containing carbohydrate mannitol and the indicator phenol red, a pH indicator for detecting acid produced by mannitol-fermenting staphylococci. Staphylococcus aureus produces yellow colonies with yellow zones, whereas other coagulase-negative staphylococci produce small pink or red colonies with no color change to the medium. If an organism can ferment mannitol, an acidic byproduct is formed that causes the phenol red in the agar to turn yellow. It is used for the selective isolation of presumptive pathogenic (pp) Staphylococcus species. MR-VP Broth: See Biochemical Tests for MR-VP Nutrient Agar Nutrient Broth Nutrient Gelatin Penassay Agar Phenyl Ethanol Agar Phenol Red Broth Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate. We use three different kinds of phenol red broths. One contains glucose; one contains lactose, and the last contains sucrose. The objective of the exercise is to determine which organisms can utilize each sugar. Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed. More in-depth information on the biochemical pathways involved in carbohydrate fermentation can be found in your lab manual. A. B. C. D. No pic A. The tube inoculated with Escherichia coliproduces acid and gas. It would be read as A/G. B. The tube inoculated with Enterococcus faecalis , produces acid but no gas. It would be read as A/-. C. The Micrococcus luteus provides no reaction and would be read as -/-. D. The Alcaligenes faecalisdegrades peptone and creates an alkaline by-product. It would be read as K. SIM agar Simmon's Citrate Agar Triple Sugar Iron Tryptic Soy Agar Tryptic Soy Agar Plus Lactose Urea broth